Study of protein interactions is vital to an understanding of many biological processes, such as the roles of gene products in vivo both in health and disease. Peptide aptamers in particular have emerged as important molecular tools that are useful for both basic and applied aspects of molecular medicine. Due to their ability to specifically bind to, and inactivate, a given target protein at the intracellular level, they provide an experimental strategy for functional protein analyses, both in vitro and in vivo. They may also be used against extracellular proteins. As well as applications in studying protein function, these tools may therefore be useful for molecular detection, diagnostics and/or as therapeutic agents. Peptides and peptide aptamers may be used free in solution. However, small peptides when unconstrained will tend to form structures which present a limited interaction surface. Furthermore, they will often lose conformational entropy upon association with target molecules, reducing free energy of binding and consequently free peptides will often not form tight non-covalent complexes, which is a problem. In addition, within cells peptides are rapidly degraded, which limits their effectiveness for the study of protein interactions in vivo, which is also a problem.
Rather than being used in free solutions, peptides of interest may be bound to physical supports, or displayed in the context of a larger polypeptide. The former cannot readily be applied to in vivo studies. In the latter, peptides are genetically inserted into the primary sequence of a simple, stable scaffold protein. The folding of the scaffold conformationally constrains the peptide, so peptide aptamers bind partners with high specificity and affinity. It is display in the context of a polypeptide which is important in the present invention. Such display is often brought about using scaffold proteins.
Prior art scaffolds have included inactivated staphylococcal nuclease, green fluorescent protein (GFP) and thioredoxin A (TrxA), as well as isolated protein folds such as the Z domain of staphylococcal protein A, “affibodies”, anticalins, and ankyrin repeats.
Further prior art scaffold proteins include the fibronectin type III domain (‘Fn3’), lipocalin family proteins from which anticalins are derived, bilin binding protein (BBP), and others.
More recently (WO 20061131749) describes several rational mutations made in Stefin A to improve it as a scaffold. The modified Stefin A scaffold comprises mutations at the following three sites Lys71-Leu73, V48D and G4W and is referred to as STM (Stefin A Triple Mutant). It was shown that the combination of these three mutations generated a protein that had minimal interactions with proteins in human cells, and in particular had lost all detectable interaction with its known natural partners. However, we found that insertion of peptides into the protein at position 71-73 led to a strong selection pressure for truncations of the protein at the end of the inserted peptide. Although such truncated proteins could display biological efficacy, this observation leads to concerns that a subset of peptides that are simply inserted at position 71-73 without truncation may not be freely available for interaction with a target protein, which is a problem. Furthermore, insertion of peptides at a single site inevitably limits the total surface area used for a protein interaction, which in turn limits binding affinity and potentially specificity.
The novel mutations made to Stefin A and to modified artificial proteins based on Stefin A such as STM (Stefin A Triple Mutant) as disclosed in the present invention provide alternative improved and more stable scaffold proteins and also provide display systems that are more versatile than those of the prior art. Moreover, these new protein scaffolds/display systems are also quite unpredictable as efficient and robust display entities. The new mutations described hereinafter have been made at specified diverse areas of the Stefin A/STM proteins and surprisingly have been found not to affect Stefin A/STM protein configuration or their potential function as scaffold proteins. Furthermore, with the improved scaffolds of the present invention by virtue of further engineering it is possible to provide modifications wherein the scaffolds have multiple insertions something that was not hitherto possible in the prior art scaffolds.